Depolarization of an individual mitochondrion or small clusters of mitochondria within cells has been achieved using a photoactivatable probe. The probe is targeted to the matrix of the mitochondrion by an alkyltriphenylphosphonium lipophilic cation and releases the protonophore 2,4-dinitrophenol locally in predetermined regions in response to directed irradiation with UV light via a local photolysis system. This also provides a proof of principle for the general temporally and spatially controlled release of bioactive molecules, pharmacophores, or toxins to mitochondria with tissue, cell, or mitochondrion specificity.

Mitochondria are central to
eukaryotic cells. As well as consuming ∼95% of the O2 inspired to generate most of the cell’s ATP, they control
cell death, contain much of central metabolism, and modulate calcium
and redox signaling. Consequently there is considerable interest in
designing mitochondria-targeted molecules to report on and manipulate
mitochondrial function.(1) The use of lipophilic
cations such as the alkyltriphenylphosphonium (TPP) moiety has enabled
the delivery of a wide range of molecules to mitochondria without
the need to alter genes or protein structure.1,2 However,
this procedure is not selective for particular mitochondria within
an organism. The challenge remains of how to use this system to deliver
molecules to mitochondria within individual tissues or cells or to
individual mitochondria in a cell at a specific time. The latter point
is significant as the organization and localization of mitochondria
within cells is important for their function3−7 and subpopulations of mitochondria contribute differently
to cell processes depending on their location within the cell.6−11 Furthermore, temporal control is vital to understanding cellular
mechanisms, so that transient changes can be observed and causal relationships
established. To address this need we have developed a procedure to
target molecules to mitochondria by coupling them to the lipohilic
TPP cation via a photocleaveable linker,(12) enabling the release of the molecule within chosen mitochondria
at a specific moment by selective irradiation of those mitochondria.

As a first example of this class of molecule here we describe MitoPhotoDNP,
which can release a protonophore within selected mitochondria thereby
stopping ATP production in and calcium ion uptake by a chosen single
mitochondrion or localized subpopulation of mitochondria within a
cell (Figure 1).

MitoPhotoDNP has three constituent parts, a caged
protonophore,
a photocleavable linker and a mitochondria-targeting unit. We chose
to target a protonophore to mitochondria as this is a way of selectively
decreasing the mitochondrial membrane potential, which is at the heart
of mitochondrial function. To produce ATP by oxidative phosphorylation
the mitochondrial electron transport chain uses the oxidation of substrates
by oxygen to pump protons out of the matrix to generate a proton electrochemical
potential gradient across the mitochondrial inner membrane (MIM),
which is composed mainly of a membrane potential (150–170 mV,
negative inside). Protons flow down this gradient through the ATP
synthase to form ATP. Ca2+ uptake into the mitochondria
through the calcium uniporter is also driven by the membrane potential
and provides a mechanism of modulating the mitochondrial and cytosolic
Ca2+ concentration locally, which is necessary for many
cellular processes.(13) Oxidation can be
uncoupled from ATP synthesis and Ca2+ transport by dissipating
the membrane potential, typically by mildly acidic lipophilic compounds,
which act as protonophores. These protonophores, or uncouplers, are
deprotonated in the mitochondrial matrix to form lipophilic anions,
which can cross the MIM, pick up a proton, and return, so abolishing
the membrane potential and switching off mitochondrial ATP production.
The classic mitochondrial uncoupler is dinitrophenol (DNP)14−16 (pKa = 4.1(17)) and in MitoPhotoDNP DNP is caged by linking to an o-nitrobenzyl group, which is a well-tried photoactivatable linker.14,18,19 The TPP cation is included as
a mitochondria-targeting group because it easily permeates biological
membranes due to its hydrophobicity and large ionic radius, and it
accumulates several hundred fold in the mitochondrial matrix within
cells in vivo due to the large membrane potential
across the MIM, in accordance with the Nernst equation.1,2 It also has very low toxicity; indeed, a TPP-containing antioxidant,
MitoQ, has been fed long-term to both animals and humans without toxicity
at its therapeutic dose.(20)

The most
scalable and reproducible synthesis of MitoPhotoDNP involved
alkylation of a commercially available phenol 1 (Scheme 1), and then reduction of the resulting aldehyde 2 to give alcohol 3. This was converted into
the phosphonium salt 4 and the caged uncoupler introduced
to complete the synthesis of MitoPhotoDNP. UV irradiation of a solution
of MitoPhotoDNP in CDCl3 led to release of DNP, detected
by 1H NMR spectroscopy.

To assess whether MitoPhotoDNP was taken up
by mitochondria in
response to the membrane potential across the inner membrane we constructed
an electrode that responded selectively to the TPP moiety of MitoPhotoDNP.(21) When mitochondria were added in the presence
of the respiratory inhibitor rotenone to prevent development of a
membrane potential there was a decrease in the concentration of MitoPhotoDNP
due to the expected adsorption of the hydrophobic TPP compound to
mitochondrial membranes.(22) Subsequent addition
of the respiratory substrate succinate led to the rapid and extensive
accumulation of MitoPhotoDNP by energized mitochondria, and this was
reversed by addition of FCCP [carbonylcyanide 4-(trifluoromethoxy)phenylhydrazone]
to abolish the mitochondrial membrane potential.14,23 This semiquantitative approach showed that MitoPhotoDNP is rapidly
and extensively accumulated by energized mitochondria in response
to the membrane potential, as expected for a TPP cation.1,2

Next, we demonstrated that MitoPhotoDNP can be photoactivated
within
cells to uncouple selectively an individual mitochondrion or a small
group of mitochondria. Freshly isolated colonic smooth muscle cells
were loaded with the mitochondrial membrane potential-sensitive dye
tetramethylrhodamine ethyl ester (TMRE) and showed a punctate fluorescent
staining of the mitochondria, consistent with energized mitochondria
with high membrane potentials. A short (85 ms) irradiation of site
1 with UV at 355 nm in the absence of MitoPhotoDNP did not lead to
any change in the pattern of TMRE fluorescence indicating no change
in mitochondrial membrane potential in response to UV alone (Figure 2, i). There was no change when the cells were exposed
to a low concentration of MitoPhotoDNP (200 nM), indicating that MitoPhotoDNP
itself did not disrupt mitochondrial function. However, when site
2 was irradiated with UV light in the presence of MitoPhotoDNP, there
was localized loss of TMRE fluorescence (red region in panel ii).
The depolarization was maintained for the duration of the experiment,
which was typically limited to 30 min, a period during which there
was little TMRE bleaching and cell performance was unaltered. When
all of the cell’s mitochondria were depolarized with rotenone
an irreversible loss of TMRE fluorescence was observed throughout
the cell (panel iii), confirming that the depolarization caused by
MitoPhotoDNP and UV light was selective. In control experiments, smooth
muscle cells treated with 200 nM MitoPhotoDNP for the same length
of time (30 min) showed no alterations in gross cellular morphology,
basal cytosolic [Ca2+] or responsiveness to calcium-generating
agonists, and the compound was not toxic to C2C12 myoblasts following
chronic exposure (overnight) to concentrations below 5 μM.

The mitochondrial depolarization remained localized
and was not
reversed during the period for which observation of an individual
mitochondrion is feasible (up to 1 h). Further experiments confirmed
that the sustained depolarization was not the result of local depletion
of ATP or mitochondrial metabolites. Cells were patch clamped(3) in whole cell configuration and using the access
afforded by the electrode, ATP (3 mM), phosphocreatine (5 mM), malate
(2.5 mM), and pyruvate (2.5 mM) were introduced into the cell. Irradiation
of a region of the cell again resulted in depolarization that was
localized and sustained, and a flickering event(4) in the unaffected region further confirmed that if repolarization
had occurred, it would have been observed (see Supporting Information).

The several-hundred-fold accumulation
of MitoPhoto-DNP within the
mitochondrial matrix in cells is vital for the selective depolarization
of mitochondria. This is supported by the fact that addition of 200
nM DNP has no effect on mitochondrial membrane potential, with 90
μM DNP being required to decrease the ratio of fluorescence
of TMRE to Mitotracker (a mitochondrial dye whose uptake is not reversed
upon mitochondrial membrane depolarization) to half maximal within
10 min. The importance of targeting is further supported by the finding
that no UV-induced depolarization of mitochondria was observed when
an untargeted photoactivatable DNP 5 (Figure 3) was employed at a concentration of 200 nM or even
when used at a concentration of 30 μM (an observation which
is consistent with the limited effectiveness of an earlier untargeted
photoactivatable uncoupler(24)).

While UV alone does not cause depolarization of
mitochondria, it
could be suggested that the TPP targeting group or the nitroarene
trigger unit of MitoPhotoDNP, in conjunction with UV, sensitized mitochondria
to photodamage and thus led to uncoupling. This possibility was eliminated
by incubating cells with 3 μM of the TPP-targeted nitro compound 6 (Figure 3) that cannot release an
uncoupler and showing that the mitochondria were not depolarized upon
irradiation. As well as releasing DNP, irradiation of MitoPhotoDNP
will also generate TPP-conjugated side products from the caging group.
To determine whether these side products could affect mitochondrial
polarization, cells were incubated with 3 μM TPP-conjugated o-nitrobenzylic alcohol 4 and irradiation of
a small cluster of mitochondria as before gave no depolarization. o-Nitrobenzylic alcohol 4 is expected to release
water upon irradiation, and HPLC confirmed that it gives a similar
distribution of TPP-conjugated side-products to MitoPhoto-DNP under
the irradiation conditions.

In summary, in this proof of concept
study, we have shown that
we can locally activate a mitochondria-targeted compound within mitochondria
inside a cell by combining a mitochondria-targeting group with a photolyzable
linker to deliver a cargo. We showed that this effectively delivered
a protonophore to mitochondria within cells and thereby led to the
selective uncoupling of either individual or a small number of mitochondria
within a cell when used in conjunction with fluorescence imaging.
This demonstrated exquisite spatial and temporal control of mitochondrial
function. The probe itself should also allow the investigation of
the role of subpopulations of mitochondria in controlling local ATP
supply and calcium ion regulation in specific parts of cells, particularly
in highly differentiated cells such as neurons and smooth muscle cells.
In neurons, for example, mitochondria cluster at locations with high
energy demand and control subplasma membrane [Ca2+] to
manage exocytosis in nerve terminals(25) and
adrenal chromaffin cells,(26) but current
techniques that involve inhibiting mitochondrial activity throughout
the entire cell cannot elucidate the effect of the localized activity
of the organelle. Thus, we expect MitoPhotoDNP to be an important
investigational tool for relating intracellular organization of mitochondria
to complex functions. More generally, our approach could be extended
to deliver selectively a wide range of functional molecules including
specific inhibitors or activators of mitochondrial processes to study
intracellular processes and also provide a means of introducing tissue
or cell specificity to mitochondria-targeted pharmacophores not previously
achievable.

Supporting Information Available

Complete refs (2a) and (20b); experimental details
for the preparation of 4–6 and MitoPhotoDNP;
UV–vis spectra; NMR spectra showing release of DNP from MitoPhotoDNP
in response to UV irradiation; electrode responses for uptake of MitoPhoto-DNP
into isolated mitochondria; details of experiments with intact cells
(including pictures and a movie); HPLC of irradiated MitoPhotoDNP
and 4; toxicity tests; and 1H and 13C NMR spectra of all compounds synthesized. This material is available
free of charge via the Internet at http://pubs.acs.org).

C.Q. and A.G.C. acknowledge BBSRC and University of
Glasgow.
S.C., S.T.C., R.C.H., and J.G.M. acknowledge The Wellcome Trust (092292/Z/10/Z
and 092292/B/10/Z). Helena M. Cochemé is acknowledged for artwork.

Schematic representing
MitoPhotoDNP accumulation within the mitochondrial
matrix of all mitochondria and selective activation within a single
mitochondrion to depolarize the inner membrane and uncouple electron
transport from ATP synthesis and calcium transport.

[Figure ID: sch1]

Scheme 1
Synthesis of MitoPhotoDNP

[Figure ID: fig2]

Figure 2

Localized
mitochondrial depolarization following localized photolysis
of MitoPhotoDNP. Freshly isolated colonic smooth muscle cells loaded
with TMRE (10 nM) and wortmannin (10 μM, to prevent cell contraction)
display a punctate fluorescent staining (i). The cell displayed underwent
(1) a brief, localized exposure to UV laser light (355 nm for 85 ms
in region shown in panel i in the absence of MitoPhotoDNP;
(2) MitoPhotoDNP (200 nM) was washed into the cell’s bathing
medium, allowed to equilibrate for 15 min and then a second region
of the cell was exposed to UV light (85 ms, in region shown in panel
ii); (3) finally the mitochondrial inhibitors rotenone (2 μM)
plus oligomycin (3 μM) were washed into the bathing medium (panel
iii). TMRE fluorescence was detected before and after each of the
three treatments and any differences highlighted by overlaying artificially
colored images in which “before” is red and “after”
is green, such that no change results in yellow regions, loss of TMRE
staining (hence mitochondrial depolarization) results in red regions,
and any gain of fluorescence results in green. A bright-field image
of the cell plus 10 μm scale bar is shown in panel iv. This
is a typical result of more than 20 similar experiments.